668 research outputs found
Measurement of Proton Leak in Isolated Mitochondria.
Oxidative phosphorylation is an important energy-conserving mechanism coupling mitochondrial electron transfer to ATP synthesis. Coupling between respiration and phosphorylation is not fully efficient due to proton leaks. In this chapter, we present a method to measure proton leak activity in isolated mitochondria. The relative strength of a modular kinetic approach to probe oxidative phosphorylation is emphasized
Sequential Lonsdaleite to Diamond Formation in Ureilite Meteorites via In Situ Chemical Fluid/Vapor Deposition.
Ureilite meteorites are arguably our only large suite of samples from the mantle of a dwarf planet and typically contain greater abundances of diamond than any known rock. Some also contain lonsdaleite, which may be harder than diamond. Here, we use electron microscopy to map the relative distribution of coexisting lonsdaleite, diamond, and graphite in ureilites. These maps show that lonsdaleite tends to occur as polycrystalline grains, sometimes with distinctive fold morphologies, partially replaced by diamond + graphite in rims and cross-cutting veins. These observations provide strong evidence for how the carbon phases formed in ureilites, which, despite much conjecture and seemingly conflicting observations, has not been resolved. We suggest that lonsdaleite formed by pseudomorphic replacement of primary graphite shapes, facilitated by a supercritical C-H-O-S fluid during rapid decompression and cooling. Diamond + graphite formed after lonsdaleite via ongoing reaction with C-H-O-S gas. This graphite > lonsdaleite > diamond + graphite formation process is akin to industrial chemical vapor deposition but operates at higher pressure (βΌ1-100 bar) and provides a pathway toward manufacture of shaped lonsdaleite for industrial application. It also provides a unique model for ureilites that can reconcile all conflicting observations relating to diamond formation
Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria
Proton leak pathways uncouple substrate oxidation from ATP synthesis in mitochondria. These pathways are classified as basal (not regulated) or inducible (activated and inhibited). Previously it was found that over half of the basal proton conductance of muscle mitochondria was catalyzed by the adenine nucleotide translocase (ANT), an abundant mitochondrial anion carrier protein. To determine whether ANT is the unique protein catalyst, or one of many proteins that catalyze basal proton conductance, we measured proton leak kinetics in mitochondria isolated from brown adipose tissue (BAT). BAT can express another mitochondrial anion carrier, UCP1, at concentrations similar to ANT. Basal proton conductance was measured under conditions where UCP1 and ANT were catalytically inactive and was found to be lower in mitochondria from UCP1 knockout mice compared to wild-type. Ablation of another abundant inner membrane protein, nicotinamide nucleotide transhydrogenase, had no effect on proton leak kinetics in mitochondria from liver, kidney or muscle, showing that basal proton conductance is not catalyzed by all membrane proteins. We identify UCP1 as a second protein propagating basal proton leak, lending support to the hypothesis that basal leak pathways are perpetrated by members of the mitochondrial anion carrier family but not by other mitochondrial inner membrane proteins
Background risk of breast cancer and the association between physical activity and mammographic density
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A comparative analysis reveals weak relationships between ecological factors and beta diversity of stream insect metacommunities at two spatial levels
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Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells
The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΞΟM) in pancreatic Ξ²-cells. This built on our previously introduced calculation of the absolute magnitude of ΞΟM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΞΟP) indicator. ΞΟM is a central mediator of glucose-stimulated insulin secretion in pancreatic Ξ²-cells. ΞΟM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΞΟM and ΞΟP. Glucose addition caused hyperpolarization of ΞΟM and depolarization of ΞΟP. The hyperpolarization was a monophasic step increase, even in cells where the ΞΟP depolarization was biphasic. The biphasic response of ΞΟP was associated with a larger hyperpolarization of ΞΟM than the monophasic response. Analysis of the relationships between ΞΟP and ΞΟM revealed that primary dispersed Ξ²-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with Ξ²-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΞΟM and ΞΟP. Finally, we demonstrate a significant problem with using an alternative ΞΟM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited Ξ²-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusion
Misconceptions about Aerobic and Anaerobic Energy Expenditure
The measurement of gas exchange has played an invaluable role in metabolic interpretation. The uptake of 1 liter of oxygen is often converted into an energy expenditure estimate of 21.1 kilojoules (e.g., 1 L O2 = 21.1 kJ or ~5 kcal). This article demonstrates both the importance of such a conversion and the potential for misinterpretation. Oxygen uptake during heavy and severe exercise will also be discussed
Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons
Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria
Microbial Spy Games and Host Response: Roles of a Pseudomonas aeruginosa Small Molecule in Communication with Other Species
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